Estrogen Receptor alpha Monoclonal Antibody (SP1)

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Publication protocol

"Antibodies used for IHC are listed in Supplementary Table 1. All IHC was performed with formalin-fixed, paraffin-embedded tissue sections. Briefly, 5-μm-thick sections were obtained with a microtome, transferred onto adhesive slides, and dried at 62°C for 30 minutes.

Using the Discovery XT automated immunohistochemistry stainer (Ventana Medical Systems, Inc., Tucson, AZ, USA), slides were stained as the following procedure. Detection was done using the Ventana DAB Map Kit (Ventana Medical Systems).

Tissue sections were deparaffinized using EZ Prep solution. CC1 standard® (pH 8.4 buffer containing Tris/Borate/EDTA) was used for antigen retrieval. Inhibitor D® (3% H2O2, Endogenous peroxidase) was blocked for 4 min at 37°C temperature. Slides were incubated with primary antibodies (PHH3, Polyclonal, 1:100, Cell Marque, Rocklin, CA, USA; anti-Ki67 antibodies, clone MIB-1, DAKO, Glostrup, Denmark) for 40 min at 37°C, and a secondary antibody of biotinylated anti-mouse immunoglobulin for 20 min at 37°C. Slides were incubated in SA-HRP D® (peroxidase-labeled streptavidin using a labeled streptavidin biotin kit) for 16 min, at 37°C and then 3,3′-diaminobenzidine chromogen combined H2O2 substrate for 8 min followed by Harris hematoxylin and bluing reagent counterstain at 37°C for 4 minutes. Reaction buffer (pH 7.6 Tris buffer) was used as washing solution. Tonsilar tissue was used for both positive and negative controls."

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